There are two types of receptors for the neurotransmitter, acetylcholine: muscarinic receptors and nicotinic receptors, based on the selectivity of action of muscarine and nicotine, respectively. Muscarinic receptors are G protein-coupled receptors. Nicotinic receptors are members of the ligand-gated ion channel family. When activated, the conductance of ions across the nicotinic ion channels increases. The nicotinic α7 receptor channel is expressed in various brain regions and is believed to be involved in many important biological processes in the central nervous system (CNS), including learning and memory.
Nicotinic α7 receptor protein forms a homo-pentameric channel in vitro that is highly permeable to a variety of cations (e.g., Ca++). Each nicotinic α7 receptor protein has four transmembrane domains, named M1, M2, M3 and M4. The M2 domain has been suggested to form the wall lining the channel. Sequence alignment shows that nicotinic α7 receptor protein is highly conserved during evolution. The M2 domain that lines the channel is identical in protein sequence from chicken to human. For discussions of the nicotinic α7 receptor, see, e.g., Revah et al. (1991), Nature 353, 846-849; Galzi et al. (1992), Nature 359, 500-505; Fucile et al. (2000), PNAS 97 (7), 3643-3648; Briggs et al. (1999), Eur J Pharmacol 366 (2-3), 301-308; and Gopalakrishnan et al. (1995), Eur J Pharmacol 290 (3), 237-246.
Fluorometric imaging is a technique that utilizes the fluorescent signal of Ca++-sensitive dyes to detect changes in intracellular Ca++. Technology is available to measure cell signals in, e.g., 96 and 384-well formats. One such technology is provided by Molecular Devices Corporation and is called Fluorometric Imaging Plate Reader, or FLIPR. FLIPR allows for high throughput imaging assays, simultaneously detecting Ca++ signals from cells, providing real time kinetic data in, e.g., a 96- or 384-well format.
Historically, detection of Ca++ signals using nicotinic receptors has been done on isolated cells. Cells transfected with either wild-type or mutant α7 receptor may exhibit changes in Ca++ as detected by fluorometric imaging using a fluorescent microscope. In addition, alpha 7 receptors have been screened in a Xenopus oocyte expression system or in stable cell lines (e.g. HEK-293) expressing the α7 receptor, using electrophysiological techniques such as a patch clamp to measure the membrane potential change during depolarization of the channel. Patch-clamp analysis is time consuming and very low throughput. It is not useful for screening compound libraries.